Peptides for the treatment of resorptive bone disease

ABSTRACT

Described are methods and associated uses for the treatment of resorptive bone disease using peptides comprising all of part of the C-terminal portion of soricidin. Also described are methods and associated uses for inhibiting osteoclast activity and/or bone resorption using the peptides.

FIELD OF THE INVENTION

The present invention relates to the treatment of bone disease, and morespecifically to peptides for the treatment of resorptive bone disease.

BACKGROUND OF THE INVENTION

Bone remodeling is mediated by bone-forming osteoblasts and osteoclaststhat are involved in bone resorption. Bone resorption is the process bywhich osteoclasts break down bone through the action of various enzymes,releasing minerals and calcium into the blood. Accelerated bone turnoverbrought about by increased osteoclast activity is a driving force behindage-related bone loss such as osteoporosis. Patients with multiplemyeloma also show evidence of bone disease and increased osteoclasticbone resorption. For some individuals with bone disease, the loss ofbone tissue is sufficiently great so as to cause mechanical failure ofthe bone structure and bone fractures.

Currently there are few treatment options available for resorptive bonedisease. One approach is the use of bisphosphonates, which are commonlyused for preventing the loss of bone mass and to treat osteoporosis.However, complex dosing guidelines often result in poor compliance withtreatment (Cramer et al., Clin Ther. 2006 October; 28(10):1686-94).Furthermore, oral bisphosphonates can cause upset stomach andinflammation and erosions of the esophagus, while intravenous treatmentwith bisphosphonates has been associated with osteonecrosis of the jaw(Dimopoulos et al., Haematologica. 2006 July; 91(7):968-71).

Soricidin (NCBI accession no. P0C2P6) is a fifty-four amino acidparalytic peptide isolated from the submaxilary saliva gland of theNorthern Short-tailed Shrew (Blarina brevicauda). Previous patents havedescribed isolation of the soricidin peptide and provided data showingthat the 54-mer peptide caused paralysis and inhibited calcium uptake intwo ovarian cancer cell lines (see U.S. Pat. Nos. 7,119,168 and7,273,850, incorporated by reference herein in their entirety).

Peptides corresponding to certain C-terminal sequences of soricidin havebeen shown to inhibit Transient Receptor Potential Vanilloid channel 6(TRPV6) without paralytic activity and to be useful for the treatment ofcancer, including metastatic cancer (see US Patent application no.20110071089, incorporated by reference herein in its entirety). Thepeptides maintain TRPV6 calcium channel binding activity without thesodium-channel binding paralytic activity of the full-length soricidinpeptide.

There remains a need for novel treatments for resorptive bone diseasessuch as osteoporosis.

SUMMARY

In one aspect, peptides corresponding to the C-terminal end of soricidinhave been determined to be useful for inhibiting osteoclast activity andbone resorption. SOR-C13 (KEFLHPSKVDLPR; SEQ ID NO: 1) and SOR-C27(EGKLSSNDTEGGLCKEFLHPSKVDLPR; SEQ ID NO: 2) reduced levels of boneresorption and osteoclast differentiation in vitro. Peptidescorresponding to the C-terminal end of soricidin as described herein aretherefore expected to be useful for the treatment of resorptive bonedisease and in particular diseases such as osteoporosis, Paget's diseaseof bone, bone metastases and bone disease associated with multiplemyeloma. Previously, the effect of soricidin-derived peptides onosteoclast activity and bone resorption was unknown.

Accordingly, in one aspect there is provided a method for treating orpreventing resorptive bone disease in a subject in need thereof,comprising administering to the subject a peptide comprising all or partof the amino acid sequence KEFLHPSKVDLPR (SEQ ID NO:1). Also provided isthe use of a peptide comprising all or part of the amino acid sequenceKEFLHPSKVDLPR (SEQ ID NO:1) for treating of preventing resorptive bonedisease in a subject in need thereof. Also provided is a peptidecomprising all or part of the amino acid sequence KEFLHPSKVDLPR (SEQ IDNO:1) for treating of preventing resorptive bone disease in a subject inneed thereof. Also provided are methods, peptides as described hereinand uses thereof for the treatment or prevention of myeloma bonedisease.

In one embodiment, the peptides described herein are useful for treatingor preventing resorptive bone disease in a subject in need thereof. Inone embodiment, the subject has a resorptive bone disease. In oneembodiment, the subject has osteoporosis, Paget's disease of bone orbone metastases. In one embodiment, the subject has resorptive bonedisease associated with multiple myeloma.

In one aspect, there is provided a method for inhibiting osteoclastactivity comprising contacting one or more osteoclasts with a peptidecomprising all or part of the amino acid sequence KEFLHPSKVDLPR (SEQ IDNO:1). Also provided is the use of a peptide comprising all or part ofthe amino acid sequence KEFLHPSKVDLPR (SEQ ID NO:1) for inhibitingosteoclast activity. Also provided is a peptide comprising all or partof the amino acid sequence KEFLHPSKVDLPR (SEQ ID NO:1) for inhibitingosteoclast activity.

In one embodiment, the osteoclasts are in vivo or in vitro. In oneembodiment, the osteoclasts are in contact with bone. In one embodiment,the osteoclasts are in contact with bone and contacting the osteoclastswith the peptide inhibits bone resorption. In one embodiment, thepeptide inhibits osteoclast differentiation. In on embodiment, theosteoclasts express TRPV6. In one embodiment, the activity of theosteoclasts has been increased by osteoclast activating factors secretedby one or more myeloma cells.

In one aspect, the peptides described herein comprise all or part of theC-terminal portion of soricidin. For example, in one embodiment thepeptide comprises at least 10 contiguous amino acids of KEFLHPSKVDLPR(SEQ ID NO: 1) or at least 10 contiguous amino acids of the C-terminalsequence of EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2). In oneembodiment, the peptide consists of at least 10 contiguous amino acidsof the C-terminal sequence of EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO:2). In one embodiment, the peptide comprises, consists essentially of,or consists of between 5 and 27 contiguous amino acids of the C-terminalsequence of EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2). In oneembodiment, the peptide comprises, consists essentially of, or consistsof at least 5, 6, 7, 8, 9, 10 or greater than 10 contiguous amino acidsof SEQ ID NO: 1 or the C-terminus of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the peptide has at least 70%, at least 75%, at least80%, at least 85%, at least 90%, or at least 95% identity toKEFLHPSKVDLPR (SEQ ID NO: 1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO:2). In one embodiment, the peptide comprises, consists essentially of,or consists of the amino acid sequence KEFLHPSKVDLPR (SEQ ID NO: 1) orEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).

In one embodiment, the peptide is a homolog, analog, mimetic, fragmentor derivative of all or part of the amino acid sequence KEFLHPSKVDLPR(SEQ ID NO: 1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).

In one embodiment, the peptide comprises all or part of the amino acidsequence KEFLHPSKVDLPR (SEQ ID NO:1) and inhibits osteoclast activity.In one embodiment, the peptide comprises all or part of the amino acidsequence KEFLHPSKVDLPR (SEQ ID NO:1) and inhibits the differentiation ofosteoclasts from cells such as bone marrow mononuclear cells and/ormacrophages. In one embodiment, the peptide comprises all or part of theamino acid sequence KEFLHPSKVDLPR (SEQ ID NO:1) and inhibits boneresorption. In one embodiment, the peptide comprises all or part of theamino acid sequence KEFLHPSKVDLPR (SEQ ID NO:1) and inhibits theactivity of Tartrate-Resistant Acid Phosphatase (TRAP) in osteoclasts.In one embodiment, the peptides comprises all or part of the amino acidsequence KEFLHPSKVDLPR (SEQ ID NO:1) and inhibits TRPV6 activity.

Also provided is the use of a peptide as described herein as ananti-resorptive agent.

Also provided is the use of a peptide comprising all or part of theamino acid sequence KEFLHPSKVDLPR (SEQ ID NO: 1) or

EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2) as described herein for thepreparation of a medicament for the treatment or prevention of bonedisease.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described in relation to thedrawings in which:

FIG. 1 shows TRPV6 protein expression in human osteoclasts and myelomacells. FIG. 1A shows hematoxylin and eosin stained osteoclasts in arepresentative human bone marrow section. FIG. 1B shows TRPV6immunohistochemistry staining and a strong expression of TRPV6 proteinsin osteoclasts. FIG. 1C shows TRPV6 expression observed in both myelomacells and osteoclasts.

FIG. 2 shows TRPV6 protein expression in primary human osteoclastcultures. Proteins were extracted from undifferentiated BMCs,differentiated osteoclasts (10 ng/ml and 50 ng/ml RANKL treatedconditions) and the positive control cells, MCF-7. FIG. 2A shows a blotprobed for TRPV6 protein expression using Santacruz TRPV6 antibody. FIG.2B shows a blot probed for TRPV6 protein expression using Alomone TRPV6antibody.

FIG. 3 shows phase contrast images of in vitro generated humanosteoclasts. FIG. 3A shows control bone marrow mononuclear cells (BMC)treated with hM-CSF only. FIG. 3B shows BMCs treated with hRANKL andhM-CSF differentiated into large osteoclasts.

FIG. 4 shows phalloidin and hoechst staining of in vitro generated humanosteoclasts. FIG. 4A shows control bone marrow mononuclear cells (BMC)treated with hM-CSF only. FIG. 4B shows BMCs treated with hRANKL andhM-CSF differentiated into large, multi-nucleated osteoclasts.

FIG. 5 shows tartrate-resistant acid phosphatase (TRAP) staining of invitro generated human osteoclasts. FIG. 5A shows control bone marrowmononuclear cells (BMC) treated with hM-CSF only. FIG. 5B shows BMCstreated with hRANKL and hM-CSF differentiated into large osteoclaststhat strongly express TRAP.

FIG. 6 shows Cathepsin K protein expression by human osteoclasts invitro. Proteins extracted from undifferentiated BMCs, differentiatedosteoclasts (OCL (1) and (2) are 10 ng/ml and 50 ng/ml RANKL treatedconditions) and the positive control cells, MCF-7 were electrophoresedon a 4-20% gradient gel and transferred to PVDF membrane. Top panel isthe immunoblot shows strong expression of Cathepsin K specifically inosteoclasts; bottom panel shows MemCode protein staining.

FIG. 7A shows dose-dependent Inhibition of osteoclast activity by theTRPV6 peptide antagonist SOR-C27 (SEQ ID NO: 2) (Sor Peptide (1)) on aCorning osteoassay plate. FIG. 7B shows percentage resorption quantifiedusing ImageJ 1.48v software and statistical analyses done with GraphPadPrism 6 software. FIG. 7C shows TRAP enzyme activity in conditionedmedia of control and treated wells of the corning osteo assay platerepresented in FIG. 7A. C1=1 uM; C2=10 uM; C3=50 uM; C4=100 uM; C5=150uM.

FIG. 8A shows inhibition of osteoclast activity by TRPV6 peptideantagonists SOR-C27 and SOR-C13 (SEQ ID NO: 1) on a Corning osteoassayplate. FIG. 8B shows percentage resorption quantified using ImageJ 1.48vsoftware and statistical analyses done with GraphPad Prism 6 software.

FIG. 9 shows expression of TRPV6 in human myeloma cell lines. The toppanel shows an immunoblot showing strong TRPV6 protein expression inhuman myeloma cell lines; the bottom panel shows MemCode proteinstaining.

FIG. 10 shows the results of a 48 hr TRPV6 antagonist peptide treatmenton a human myeloma cell line (KMM-1 cells). A dose-dependent inhibitionof cell growth was observed with increasing concentrations of SOR C-13and SOR-C27. C1=1 uM; C2=10 uM; C3=50 uM; C4=100 uM; C5=150 uM; C6=250uM; C7=300 uM; C8=400 uM; C9=500 uM.

FIG. 11 shows the results of 48 hr TRPV6 antagonist peptide treatment onhuman myeloma cell line (U266 cells). A dose-dependent inhibition ofcell growth was observed with increased concentrations of SOR C-13 andSOR-C27. C1=1 uM; C2=10 uM; C3=50 uM; C4=100 uM; C5=150 uM; C6=250 uM;C7=400 uM; C8=500 uM.

DESCRIPTION OF VARIOUS EMBODIMENTS Definitions

As used herein, “resorptive bone disease” refers to any conditioncharacterized by an imbalance between osteoblast and/or osteoclastactivity leading to decreased bone strength and/or bone loss. Examplesof resorptive bone disease include, but are not limited to,osteoporosis, Paget's disease of bone, bone metastasis, and bone diseaseassociated with cancers such as multiple myeloma.

As used herein, “osteoporosis” refers to a disorder characterized by animbalance between bone resorption and bone formation leading todecreased bone strength. In one embodiment, a subject has osteoporosiswhen their bone mineral density is less than or equal to 2.5 standarddeviations below that of a young (30-40-year-old) healthy adultreference population (see e.g. WHO Scientific Group on the Preventionand Management of Osteoporosis (2000: Geneva, Switzerland) (2003). ISBN9241209216).

As used herein, “Paget's disease of bone” or “Paget's disease” refers toa chronic disorder that can result in enlarged and misshapen bonescaused by the excessive breakdown and formation of bone, followed bydisorganized bone remodeling. In one embodiment, subjects with Paget'sdisease of bone exhibit bone pain and/or elevated levels of alkalinephosphatase in the blood.

As used herein, “bone disease associated with multiple myeloma” refersto bone loss and/or bone lesions in a subject with multiple myeloma. Inone embodiment, subjects with multiple myeloma exhibit an increase inosteoclast activity caused osteoclastic activating factors released bytumor cells.

As used herein, “osteoclast” refers to a multinucleated cell capable ofresorbing mineralised bone, dentine and cartilage. In one embodiment,osteoclasts are characterized by a cytoplasm with a high concentrationof vacuoles containing lysosomes filled with acid phosphatase. In oneembodiment, osteoclasts express tartrate resistant acid phosphatase(TRAP) and cathepsin K.

As used herein “inhibiting osteoclast activity” refers to inhibiting theresorbing activity of osteoclasts and/or inhibiting the formation ofosteoclasts. In one embodiment, inhibiting osteoclast activity includesinhibiting the differentiation of macrophages and/or bone marrowmononuclear cells into osteoclasts.

Peptides for Inhibiting Osteoclasts and Treating Resorptive Bone Disease

It has surprisingly been determined that peptides corresponding to theC-terminal end of Soricidin such as SOR-C13 and SOR-C27 are useful forinhibiting osteoclast activity and bone resorption. As shown in FIGS. 7and 8, investigations into the effects of SOR-C13 and SOR-C27 using anin vitro model showed a dose-dependent decrease in bone resorption byosteoclasts. Treatment with SOR-C13 and SOR-C27 also resulted in adecrease in TRAP enzyme activity relative to controls. A number ofresorptive bone diseases such as osteoporosis, Paget's disease of boneand bone disease associated with cancers are characterized by anincrease in osteoclast activity such that inhibition of osteoclastactivity is expected to be useful for the treatment or prevention ofresorptive bone disease.

The present description therefore provides methods and uses for thetreatment or prevention of bone disease in a subject in need thereof.Also provided are methods and uses for inhibiting osteoclast activity.In one embodiment, there is provided a method for treating or preventingresorptive bone disease comprising administering to the subject apeptide comprising, consisting essentially of, or consisting of all orpart of KEFLHPSKVDLPR (SEQ ID NO: 1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQID NO: 2). Also provided is the use of a peptide comprising, consistingessentially of, or consisting of all or part of KEFLHPSKVDLPR (SEQ IDNO: 1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2) for the treatmentor prevention of resorptive bone disease in a subject in need thereof.Also provided is a peptide comprising, consisting essentially of, orconsisting of all or part of KEFLHPSKVDLPR (SEQ ID NO: 1) orEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2) for the treatment orprevention of resorptive bone disease in a subject in need thereof. Inone embodiment, the peptides described herein are useful for thetreatment, cure prevention or suppression of symptoms associated withresorptive bone disease.

Also provided are methods for inhibiting osteoclast activity comprisingcontacting one or more osteoclasts with a peptide comprising, consistingessentially of, or consisting of all or part of the amino acid sequenceKEFLHPSKVDLPR (SEQ ID NO:1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO:2). Also provided is the use of a peptide comprising, consistingessentially of, or consisting of all or part of the amino acid sequenceKEFLHPSKVDLPR (SEQ ID NO:1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO:2) for inhibiting osteoclast activity. Also provided is a peptidecomprising, consisting essentially of, or consisting of all or part ofthe amino acid sequence KEFLHPSKVDLPR (SEQ ID NO:1) orEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2) for inhibiting osteoclastactivity. Optionally, the osteoclasts are in vivo, ex vivo or in vitro.

In one embodiment, the peptide comprises from 5 to 27 contiguous aminoacids of SEQ ID NO: 2. In one embodiment, the peptide comprisescontiguous amino acids of SEQ ID NO: 2 starting from the N-terminalamino acid of SEQ ID NO: 2. In another embodiment, the peptide comprisescontiguous amino acids starting from the C-terminal sequence of SEQ IDNO: 2. In one embodiment, the peptide comprises at least 5, 6, 7, 8, 9,10 or greater than 10 contiguous amino acids of SEQ ID NO:2. Optionallythe peptide comprises at least: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 amino acids of SEQ ID NO: 2. In one embodiment, the peptidesdescribed herein comprise, consist essentially of, or consist of theamino acid sequence KEFLHPSKVDLPR (SOR-C13) orEGKLSSNDTEGGLCKEFLHPSKVDLPR (SOR-C27).

In some embodiments, amino acids may be added to the peptides describedherein. One can readily make a longer peptide by adding a variety ofadditional amino acids to the SOR-C27 sequences to make a peptide thatcould be up to, for example, 30, 35, 40 or 45 amino acids long. (e.g.additional amino acids corresponding to the soricidin amino acidsequence such as one or more of the amino acids that are immediatelytowards the N-terminal segment of SOR-C27 in soricidin(SILARPAELNTETCILEC; SEQ ID NO: 1), a targeting sequence, or other aminoacids) or longer. In one embodiment, the peptide described hereinconsists of 30 or fewer, 27 or fewer, 25 or fewer, 20 or fewer, 15 orfewer, 13 or fewer, 11 or fewer, 9 or fewer, or 7 or fewer amino acids.

The peptides described herein optionally include analogs of theaforementioned peptides. Analogs of the peptides of the inventionoptionally include, but are not limited to an amino acid sequencecontaining one or more amino acid substitutions, insertions, deletionsand/or mutations. Amino acid substitutions may be of a conserved ornon-conserved nature. Conserved amino acid substitutions involvereplacing one or more amino acids of the peptides of the invention withamino acids of similar charge, size, and/or hydrophobicitycharacteristics. When only conserved substitutions are made, theresulting analog should be functionally equivalent. Non-conservedsubstitutions involve replacing one or more amino acids of the aminoacid sequence with one or more amino acids that possess dissimilarcharge, size, and/or hydrophobicity characteristics. The analog isoptionally a peptoid, which is an N-substituted polyglycine with aminoacid R groups attached at the N atom. Another analog is optionally apeptide synthesized from D-amino acids rather than the natural L-aminoacids.

One or more amino acid insertions are optionally introduced into thepeptides described herein such as SOR-C13 or SOR-C27. Amino acidinsertions may consist of single amino acid residues or sequential aminoacids ranging for example from 2 to 15 amino acids in length.

Deletions consist of the removal of one or more amino acids, or discreteportions from the amino acid sequence of the peptide. The deleted aminoacids may or may not be contiguous.

Analogs of a peptide of the invention are optionally prepared byintroducing mutations in a nucleotide sequence encoding the peptide.Mutations in nucleotide sequences constructed for expression of analogsof a protein of the invention preserve the reading frame of the codingsequences. Furthermore, the mutations will preferably not createcomplementary regions that could hybridize to produce secondary mRNAstructures such as loops or hairpins, which could adversely affecttranslation of the mRNA.

Mutations are optionally introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures are employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a peptide of the invention is alsoreadily achieved by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA re-ligated. Exemplary methods of makingthe alterations set forth above are disclosed by Sambrook et al.(Sambrook J et al. 2000. Molecular Cloning: A Laboratory Manual (ThirdEdition), Cold Spring Harbor Laboratory Press).

In addition, peptides and/or analogs useful for the purposes of thepresent invention are readily prepared by chemical synthesis usingtechniques well known in the chemistry of proteins such as solid phasesynthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) orsynthesis in homogenous solution (Houbenweyl, 1987, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). Thepeptides of the invention also include those having sequence identity toa peptide of the invention, mutated peptides and/or truncations thereofas described herein.

Other peptides suitable for the methods and uses of the presentinvention optionally comprise, consist essentially of or consist of anamino acid sequence with at least: 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90% or 95% sequence identity to all or part of SEQ ID NO:1 or SEQID NO:2 described herein that inhibit osteoclast activity and/ordecrease bone resorption levels. In one embodiment, the peptide has atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% or atleast 95% sequence identity to the amino acid sequence KEFLHPSKVDLPR(SOR-C13) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SOR-C27). Sequence identity istypically assessed by the BLAST version 2.1 program-advanced search(parameters as above; Altschul, S. F., Gish, W., Miller, W., Myers, E.W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol.Biol. 215:403-410). BLAST is a series of programs that are availableonline through the U.S. National Center for Biotechnology Information(National Library of Medicine Building 38A Bethesda, Md. 20894) Theadvanced Blast search is set to default parameters. References for theBlast Programs include: Altschul, S. F., Gish, W., Miller, W., Myers, E.W. & Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol.Biol. 215:403-410; Gish, W. & States, D. J. (1993) “Identification ofprotein coding regions by database similarity search.” Nature Genet.3:266-272.; Madden, T. L., Tatusov, R. L. & Zhang, J. (1996)“Applications of network BLAST server” Meth. Enzymol. 266:131-141;Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs.” Nucleic Acids Res.25:3389-3402); Zhang, J. & Madden, T. L. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation.” Genome Res. 7:649-656).

In one embodiment, peptides suitable for the methods and uses ofdescribed herein are peptide mimetics. In one embodiment, the peptidemimetics are based on all or part of KEFLHPSKVDLPR (SEQ ID NO: 1) orEGKLSSNDTEGGLCKEFLHPSKVDLPR (SOR-C27). “Peptide mimetics” are structureswhich serve as substitutes for peptides in interactions betweenmolecules (See Morgan et al. (1989), Ann. Reports Med. Chem. 24:243-252for a review). Peptide mimetics include synthetic structures whichoptionally contain amino acids and/or peptide bonds but retain thestructural and functional features of a peptide, or enhancer orinhibitor of the invention. Peptide mimetics also include peptoids,oligopeptoids (Simon et al. (1972) Proc. Natl. Acad, Sci USA 89:9367);and peptide libraries containing peptides of a designed lengthrepresenting all possible sequences of amino acids corresponding to apeptide of the invention.

Peptide mimetics are designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptidesmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

In one embodiment, the peptides described herein are for use oradministered to a subject for the treatment of resorptive bone diseaseand/or to inhibit osteoclast activity. In one embodiment, the peptidesdescribed herein are for use or administered to a subject to reduceosteoclast formation.

The term “subject” as used herein includes all members of the animalkingdom and is preferably a mammal, such as human. Administering apeptide to a subject includes both in vivo and ex vivo administrations.In a preferred embodiment, the subject is a human. In one embodiment,the subject has osteoporosis, Paget's disease of bone, or bone diseaseassociated with cancer. In one embodiment, the subject experiencesnegative side-effects from treatment with commonly used drugs for thetreatment of bone disease, such as bisphosphonates.

As used herein, and as well understood in the art, “treating” or“treatment” is an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, alleviation or amelioration of one ormore symptoms or conditions, diminishment of extent of disease,stabilized (i.e. not worsening) state of disease or disorder, preventingspread of disease or disorder, delay or slowing of disease or disorderprogression, amelioration or palliation of the disease or disorderstate, and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. In one embodiment,treatment refers to reducing bone resorption in a subject with bonedisease such as to reduce or eliminate any undesirable physiologicaleffects associated with resorptive bone disease such as bone pain. Inone embodiment, treatment of subjects in need thereof with the peptidesdescribed herein may result in a reduced risk of bone fractures, reducedneed for bone surgery, reduced need for bone radiotherapy, a reducedoccurrence of spinal cord compression and/or hypercalcemia.

Optionally the peptides described herein can be formulated into apharmaceutical composition for administration to subjects in abiologically compatible form suitable for use in vivo. The term“biologically compatible form suitable for administration in vivo”refers to a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. The peptides may beadministered to living organisms including humans and animals.

Administration of a therapeutically active amount of the peptides orpharmaceutical compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof a substance may vary according to factors such as the disease state,age, sex, and weight of the individual, and the ability of the substanceto elicit a desired response in the individual. Dosage regimes may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

The peptides of the invention are preferably combined with othercomponents such as a carrier in a composition such as a pharmaceuticalcomposition or cosmetic composition. The compositions are useful whenadministered in methods for the treatment or prevention of bone disease.Optionally, the peptides described herein are administered withtherapeutic compounds or other substances that are known to cause bonedisease to moderate bone resorption such as, but not limited to,corticosteroids and/or estrogen- or testosterone-lowering therapies.

The peptides or pharmaceutical compositions can be administered tohumans or animals by a variety of methods including, but not restrictedto topical administration, oral administration, aerosol administration,intratracheal instillation, intraperitoneal injection, injection intothe cerebrospinal fluid, intravenous injection and subcutaneousinjection. Dosages to be administered depend on patient needs, on thedesired effect and on the chosen route of administration. Nucleic acidmolecules encoding for the peptides and polypeptides described hereinmay be introduced into cells using in vivo delivery vehicles such asliposomes, They may also be introduced into these cells using physicaltechniques such as microinjection and electroporation or chemicalmethods such as coprecipitation or using liposomes.

The pharmaceutical compositions are prepared by known methods for thepreparation of pharmaceutically acceptable compositions which can beadministered to patients, and such that an effective quantity of thepeptide is combined in a mixture with a pharmaceutically acceptablevehicle. Suitable vehicles are described, for example in Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., USA) or Handbook of PharmaceuticalAdditives (compiled by Michael and Irene Ash, Gower Publishing Limited,Aldershot, England (1995). On this basis, the compositions include,albeit not exclusively, solutions of the substances in association withone or more pharmaceutically acceptable vehicles or diluents, and may becontained in buffered solutions with a suitable pH and/or be iso-osmoticwith physiological fluids. In one embodiment, there is provided acomposition comprising a peptide as described herein for reducing boneresorption and/or osteoclast activity and a pharmaceutically acceptablevehicle or diluent.

The following examples illustrate embodiments of the invention and donot limit the scope of the invention.

EXAMPLES

Materials & Methods

In-Vitro Generation of Human Osteoclasts

Osteoclasts were generated in vitro by the modified method of Cody etal., 2011, using fresh human bone marrow aspirates. Briefly, bone marrowmononuclear cells (BMC) separated on a density gradient medium inSepMate tubes were resuspended in growth media (α-MEM with 10% FBS) andcultured for 3 days in T75 flask with 35 ng/mL M-CSF at 37° C. in a 5%CO2 incubator. For osteoclast differentiation experiments, 10⁵cells/well of BM monocytes-macrophage precursors seeded in a 24 wellplate was treated with human M-CSF and RANKL, and media replenished withfresh cytokines every third day for two weeks.

Osteoclast Characterization

Osteoclasts were fixed and stained for tartrate-resistant acidphosphatase (TRAP) using the leukocyte acid phosphatase kit(Sigma-Aldrich) according to the manufacturer's protocol. Images wereacquired in EVOS brightfield microscope. To visualize actin ringformation in osteoclasts, tetramethyl rhodamine-phalloidin (LifeTechnologies Inc.) staining was done for 1 hour. The cell nuclei werecounterstained with 1 μg/ml Hoechst 33342 (Sigma-Aldrich) in phosphatebuffered saline for 5 minute. Images were acquired in EVOS fluorescencemicroscope. TRAP enzyme activity was measured at 37° C. with a substratebuffer (125 mg/ml Naphthol AS-BI phosphoric acid in diazotized fastgarnet base chemical solution, Sigma-Aldrich) at 560 nm.

Western Blotting

Proteins were extracted using RIPA buffer (50 mM Tris HCL pH 7.4, 150 mMNaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM PMSF), sonicated for 15 s at30% amplitude, incubated in ice for 30 min followed by centrifugation at4° C. for 30 min at 1200 g. Protein concentration in supernatants wasquantified using Pierce™ BCA Protein Assay kit (Thermo Scientific, IL,USA). 25 μg of protein extracts mixed with Laemmli sample buffer with 5%β-mercapto ethanol and boiled for 5 min at 95° C. were resolved bySDS-PAG electrophoresis on mini-Protean™ TGX Stain-Free® any kD gel,subsequently transferred onto PVDF membrane by semi dry method usingTrans-Blot® Turbo™ Transfer system (25 Volts (V), 10 minute transfer).The membrane was then washed with TBS-T buffer (Tris.HCl, pH 7.5, 140 mMNaCl, and 0.05% Tween 20), blocked in 5% skimmed milk for 1 h andincubated overnight at 4° C. with specific primary antibody such asanti-rabbit TRPV6 Ab (Alomone, ACC-036, 1:500; and Santacruz, H-90,1:500) or Cathepsin K (Abcam, 1:200). Proteins in immunoblots weredetected with Amersham™ ECL™ Prime western blotting detection kit andimaged in a ChemiDoc™ MP imaging system (Bio-Rad Technology, CA, USA)with the Image Lab 5.0 software.

Immunohistochemistry

Paraffin embedded human bone specimen was sectioned at 5 μm thickness,decalicified and subjected to automated IHC staining in VentanaBenchmark Ultra Platform. Heat induced epitope retrieval was done withhigh pH solution for 36 min, followed by anti-TRPV6 primary antibodyincubation (Alomone ACC-036; 1:200) for 32 min. Following diaminobenzidine detection, sections were counterstained with hematoxylin.Images were acquired in Leica Microscope with LAS 4.6 Software.

Osteoclast Differentiation and Osteoassay

10⁵ BMCs per well in 24 well Corning osteoassay plate were treated withhuman M-CSF (10 ng/mL) and 10 ng/mL of human RANKL. Undifferentiatedcondition includes cells treated with M-CSF only. The treatment wellshad both M-CSF and RANKL followed by addition of TRPV6 peptideantagonists SOR-C13 and SOR-C27 at concentrations range 1 μM-150 μMtested in triplicate wells, vehicle or PBS buffer in which the peptidesare dissolved and the standard anti-resorbing agent, Zoledronate wereincluded for the experiment. Replenishment with fresh media containingM-CSF, RANKL and SOR peptides in respective wells was carried out everythird day. On day 12, cells in osteoplates were removed by incubatingwith 10% bleach for 10 minutes; wells were washed with distilled waterthree times and air-dried for 3-5 hrs. Osteoassay plate was scannedusing ChemiDoc MP and area of resorption pits in each well wasquantified using ImageJ 1.48v Software.

Statistics

Experiments were done in triplicates for each condition and datapresented as mean±SD. All statistical analyses were done using GraphPadPrism 6, one way ANOVA and Dunnett's multiple comparison test was usedto determine significance between groups and P<0.05 is consideredstatistically significant.

Expression and Localization of TRPV6 in Osteoclasts

Strong expression of TRPV6 in human osteoclasts was found byimmunohistochemical staining (FIG. 1). Osteoclasts were generated invitro by the modified method of Cody et al., 2011, using fresh bonemarrow (BM) aspirates. BM aspirates and cultured bone marrow mononuclearcells were processed for two weeks by treatment with two key cytokineshuman M-CSF and RANKL essential for osteoclast differentiation.

Osteoclast formation was observed from the sixth day onwards. The cellsthus generated in vitro exhibited phenotypic characteristics ofosteoclasts such as being large sized, multi-nucleated cells with actinrings. This is evident from the phase contrast image and the phalloidin& hoechst stained, fluorescence microscope images of osteoclasts (FIGS.3b and 4b ). These osteoclasts were found to strongly express boneresorption markers such as Tartarate-resistant acid phosphatase (FIG.5), Cathepsin K (FIG. 6), also exhibit their characteristic functionalactivity of resorbing osteoassay surface (FIG. 7). Furthermore, theosteoclasts generated from primary human bone marrow expressed TRPV6protein as determined by western blotting (FIG. 2).

Effect of C-Terminal Soricidin Peptides on Osteoclasts

The effect of C-terminal Soricidin peptides on osteoclast activity andbone resorption was examined using 24-well corning assay plates thathave a 3D osteo surface that mimics in vivo bone for in vitro bone cellassays. The inorganic bone biomaterial surface in each well is capableof supporting the functional properties of osteogenic cells. Onlyosteoclasts, specialized cells expressing bone-resorbing enzymes such asTRAP, cathepsin K and collagenase, can resorb such an artificialosteosurface. Resorption of the osteosurface is evident from theresorption pits/trails made by the osteoclasts, and the extent ofresorption was quantified. Furthermore, the percentage activity of TRAP,one of the enzymes involved in bone resorption, was determined in tandemwith the resorption assay (FIG. 7c ). The anti-resorptive potential ofselective TRPV6 antagonist peptides, SOR-C13 and SOR-027 at variousconcentrations was observed (FIGS. 7 and 8) was observed. At aconcentration of 150 μM (C5) a consistent, significant effect wasobserved where the peptides inhibited osteoclast differentiation invitro, and significantly reduced their resorptive activity on corningosteoassay surface.

The expression of TRPV6 protein was also observed in human myeloma cellsby immunohistochemistry on myeloma bone sections (FIG. 10) and inmyeloma cell lines by western blotting (FIG. 9). Moreover, the growthinhibitory potential of SOR-C13 and SOR-027 was determined using humanmyeloma cell lines U266 and KMM-1 (FIGS. 10 and 11). There exists areciprocal relationship between myeloma cells and osteoclasts in thebone marrow milieu where myeloma cells secrete many factors that promoteosteoclast activity, and in turn osteoclasts induce myeloma cell growthand survival. Hence, along with the anti-resorptive property, themyeloma cell growth inhibitory property of SOR-C13 and SOR-C27 can beapplied for treating myeloma bone disease.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

The invention claimed is:
 1. A method for treating or preventingresorptive bone disease in a human subject in need thereof, comprisingadministering to the subject a peptide comprising the amino acidsequence KEFLHPSKVDLPR (SEQ ID NO:1).
 2. The method of claim 1, whereinthe peptide consists of the amino acid sequence ofEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).
 3. The method of claim 1,wherein the peptide consists of the amino acid sequence KEFLHPSKVDLPR(SEQ ID NO: 1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).
 4. Themethod of claim 1, wherein the peptide inhibits osteoclast activity. 5.The method of claim 1, wherein the peptide inhibits bone resorption. 6.The method of claim 1, wherein the subject has osteoporosis, Paget'sdisease of bone or bone metastases.
 7. The method of claim 1, whereinthe subject has resorptive bone disease associated with multiplemyeloma.
 8. A method for inhibiting osteoclast activity comprisingcontacting one or more osteoclasts with a peptide comprising the aminoacid sequence KEFLHPSKVDLPR (SEQ ID NO:1).
 9. The method of claim 8,wherein the peptide consists of the amino acids sequenceEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).
 10. The method of claim 8,wherein the peptide comprises the amino acid sequenceEGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).
 11. The method of claim 8,wherein the peptide consists of the amino acid sequence KEFLHPSKVDLPR(SEQ ID NO: 1) or EGKLSSNDTEGGLCKEFLHPSKVDLPR (SEQ ID NO: 2).
 12. Themethod of claim 8, wherein the osteoclasts are in vivo or in vitro. 13.The method of claim 8, wherein the osteoclasts are in contact with boneand contacting the osteoclasts with the peptide inhibits boneresorption.
 14. The method of claim 8, wherein the peptide inhibitsosteoclast differentiation.
 15. The method of claim 1, wherein theosteoclasts express Transient Receptor Potential Vanilloid channel 6.16. The method of claim 8, wherein the osteoclasts activity has beenincreased by osteoclast activating factors secreted by one or moremyeloma cells.